Therapeutic benzothiazole compounds

ABSTRACT

Compounds of the formula (I)  
                 
for use as an estrogen receptor -β-selective ligand are described wherein:  
     X is O or S; and R 1 , R 3 -R 6  are as described in the specification. The use of these compounds in treating Alzheimer&#39;s disease, anxiety disorders, depressive disorders, osteoporosis, cardiovascular disease, rheumatoid arthritis and prostate cancer is described; as are processes for making them.

This application is a divisional of U.S. application Ser. No.10/450,927, which is the National Stage of International Application No.PCT/SE01/02855, filed Dec. 19, 2001.

TECHNICAL FIELD

The present invention is directed to a series of ligands, and moreparticularly to estrogen receptor-β ligands which have betterselectivity than estrogen for the estrogen receptor-β over the estrogenreceptor-α, as well as to methods for their production and use in thetreatment of diseases related to the estrogen receptor-β, specifically,Alzheimer's disease, anxiety disorders, depressive disorders,osteoporosis, cardiovascular disease, rheumatoid arthritis, or prostatecancer.

BACKGROUND

Estrogen-replacement therapy (“ERT”) reduces the incidence ofAlzheimer's disease and improves cognitive function in Alzheimer'sdisease patients (Nikolov et al. Drugs of Today, 34(11), 927-933(1998)). ERT also exhibits beneficial effects in osteoporosis andcardiovascular disease, and may have anxiolytic and anti-depressanttherapeutic properties. However, ERT shows detrimental uterine andbreast side effects that limit its use.

The beneficial effects of ERT in post-menopausal women is echoed bybeneficial effects of estrogen in models relevant to cognitive function,anxiety, depression, bone loss, and cardiovascular damage inovariectomized rats. Estrogen also produces uterine and breasthypertrophy in animal models reminiscent of its mitogenic effects onthese tissues in humans. Specifically, experimental studies havedemonstrated that estrogen effects the central nervous system (“CNS”) byincreasing cholinergic function, increasing neurotrophin/neurotrophinreceptor expression, altering amyloid precursor protein processing,providing neuroprotection against a variety of insults, and increasingglutamatergic synaptic transmission, among other effects. The overallCNS profile of estrogen effects in pre-clinical studies is consistentwith its clinical utility in improving cognitive function and delayingAlzheimer's disease progression. Estrogen also produces mitogeniceffects in uterine and breast tissue indicative of its detrimental sideeffects on these tissues in humans.

The estrogen receptor (“ER”) in humans, rats, and mice exists as twosubtypes, ER-α and ER-β, which share about a 50% identity in theligand-binding domain (Kuiper et al. Endocrinology 139(10) 4252-4263(1998)). The difference in the identity of the subtypes accounts for thefact that some small compounds have been shown to bind preferentially toone subtype over the other (Kuiper et al.).

In rats, ER-β is strongly expressed in brain, bone and vascularepithelium, but weakly expressed in uterus and breast, relative to ER-α.Furthermore, ER-α knockout (ERKO-α) mice are sterile and exhibit littleor no evidence of hormone responsiveness of reproductive tissues. Incontrast, ER-β knockout (ERKO-β) mice are fertile, and exhibit normaldevelopment and function of breast and uterine tissue. Theseobservations suggest that selectively targeting ER-β over ER-α couldconfer beneficial effects in several important human diseases, such asAlzheimer's disease, anxiety disorders, depressive disorders,osteoporosis, and cardiovascular disease without the liability ofreproductive system side effects. Selective effects on ER-β-expressingtissues (CNS, bone, etc.) over uterus and breast could be achieved byagents that selectively interact with ER-β over ER-α.

It is a purpose of this invention to identify ER-β-selective ligandsthat are useful in treating diseases in which ERT has therapeuticbenefits.

It is another purpose of this invention to identify ER-β-selectiveligands that mimic the beneficial effects of ERT on brain, bone andcardiovascular function.

It is another purpose of this invention to identify ER-β-selectiveligands that increase cognitive function and delay Alzheimer's diseaseprogression.

SUMMARY OF THE INVENTION

This present invention is directed to compounds having the genericstructure:

These compounds are ER-β-selective ligands, which mimic ERT, but lackundesirable side effects of ERT and are useful in the treatment orprophylaxis of Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritis orprostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of absorbance values obtained in assays for thebinding to the estrogen receptor of the ER agonist 17-β-estradiol (E)and the ER antagonist ICI182,780 (A) for cells transfected either withαER or βER.

FIG. 2 shows typical concentration-response curves, providing EC₅₀values, for binding to αER and βER.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of the formula (I)

for use as ER-β-selective ligands:

wherein:

X is O or S;

R¹ is C₁₋₈alkyl, phenyl, benzyl or a 5- or 6-membered ring heterocyclecontaining 1, 2 or 3 heteroatoms each independently selected from O, Nand S and additionally having 0 or 1 oxo groups and 0 or 1 fused benzorings, wherein the C₁₋₈alkyl, phenyl, benzyl or heterocycle issubstituted by 0, 1, 2 or 3 substituents selected from -R^(a), —OR^(a),SR^(a), NR^(a)R^(a), CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl;

R³ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R, —OC(═O)R^(a),—C(═O)NR^(a)R^(a), NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl; or R³ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro;

R⁴ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl;

R⁵ is —R, —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl;

R⁶ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl; or R⁶ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro; and

R^(a) is H, C₁₋₆alkyl, C₁₋₃haloalkyl, phenyl or benzyl;

and pharmaceutically acceptable salts thereof.

In the above definitions, where R^(a) appears twice in a group, each maybe separately selected from the possible values.

These compounds are useful in treating disease conditions related to theβ-estrogen receptor, more particularly in treating Alzheimer's disease,anxiety disorders, depressive disorders, osteoporosis, cardiovasculardisease, rheumatoid arthritis and prostate cancer.

In another aspect the present invention provides the use of a compoundof the formula (I) or a pharmaceutically acceptable salt thereof for themanufacture of a medicament for the treatment (including prophylaxis) ofdisease conditions related to the β-estrogen receptor, more particularlyin treating Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritisand prostate cancer.

In a further aspect the present invention provides a method of treatingdisease conditions related to the β-estrogen receptor, more particularlyin treating Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritisand prostate cancer.

In one embodiment R¹ is C₁₋₈alkyl, phenyl, benzyl or a 5- or 6-memberedring heterocycle containing 1, 2 or 3 heteroatoms each independentlyselected from O, N and S and additionally having 0 or 1 oxo groups and 0or 1 fused benzo rings, wherein the C₁₋₈alkyl, phenyl, benzyl orheterocycle is substituted by 0, 1, 2 or 3 substituents selected from—R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl.

In another embodiment R³ is C₁₋₆alkyl, —OR^(a), —SR^(a), —NR^(a)R^(a),—CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R, —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; or R³ is C₁₋₃alkylcontaining 1 or 2 substituents selected from —OR^(a), —SR^(a),—NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano and nitro.

In another embodiment R⁴ is —R^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl.

In another embodiment R⁵ is —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl.

In another embodiment R⁶ is C₁₋₆alkyl, —OR^(a), —SR^(a), —NR^(a)R^(a),—CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; or R⁶ isC₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a), —SR^(a),—NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano and nitro.

In another embodiment R¹ is phenyl or benzyl, wherein the phenyl orbenzyl is substituted by 0, 1, 2 or 3 substituents selected from —R^(a),—OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl. In a more specific embodiment, R¹ is4-hydroxyphenyl substituted by 0, 1 or 2 substituents selected from—R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl.

In one aspect X is S. In another aspect X is O.

R¹ may be a 5- or 6-membered ring heterocycle, unsubstituted orsubstituted as defined hereinabove; for example the 5- or 6-memberedring may be thiophene, furan, pyrrolidinone, pyridine, indazole orthiazolidinone. In a preferred aspect R¹ is phenyl unsubstituted orsubstituted as defined hereinabove. Examples of R¹ being substitutedphenyl include hydroxyphenyl (for example 4-hydroxyphenyl or3-hydroxyphenyl), C₁₋₄alkoxyphenyl (for example 4-methoxyphenyl or3-methoxyphenyl), halophenyl (for example bromophenyl such as2-bromophenyl or chlorophenyl such as 2-chlorophenyl), C₁₋₄alkylphenyl(for example methylphenyl such as 2-methylphenyl or 3-methylphenyl orethylphenyl such as 2-ethylphenyl or propylphenyl such as2-isopropylphenyl), cyanophenyl (for example 2-cyanophenyl) ortrifluoromethylphenyl (for example 4-trifluoromethylphenyl).

In particular R¹ is hydroxyphenyl.

In a particular aspect R³is halo, cyano, carbamoyl or C₁₋₆alkyl; moreparticularly halo for example chloro or bromo, cyano, or C₁₋₆ alkyl forexample methyl or ethyl. In another particular aspect R³ is hydrogen.

In a particular aspect R⁴ is halo, for example chloro or bromo, hydroxyor C₁₋₆ alkoxy, for example methoxy or ethoxy; more particularly R⁴ ishydroxy or methoxy, for example hydroxy. In another particular aspect R⁴is hydrogen.

In a particular aspect R⁵ is halo, for example chloro or bromo, hydroxyor C₁₋₆ alkoxy, for example methoxy or ethoxy; more particularly R⁵ ishydroxy or methoxy, for example hydroxy. In another particular aspect R⁵is hydrogen.

In a particular aspect R⁶ is halo, for example chloro or bromo,C₁₋₄alkyl for example, methyl or ethyl, trifluoromethyl, hydroxy,C₁₋₄alkoxy, for example methoxy or ethoxy, carboxy, C₁₋₄alkoxycarbonyl,for example methoxycarbonyl, cyano, halomethyl, for example bromomethyl,cyanoC₁₋₄alkyl, for example cyanomethyl, carbamoyl, methylcarbamoyl ordimethylcarbamoyl. In another particular aspect R⁶ is hydrogen. In oneembodiment R⁶ is halo, cyano or C₁₋₆alkyl.

Preferred benzoxazoles are those wherein R¹ is 4-hydroxyphenyl or3-chloro-4-hydroxy phenyl; R³ is chloro or bromo; R⁵ is hydroxy; and R⁴and R⁶ are both hydrogen.

Preferred benzthiazoles are those wherein R¹ is 4-hydroxyphenyl; R⁶ iscyano or carboxy; R⁴ is hydroxy; and R³ and R⁵ are both hydrogen.

Compounds within the formula (I) have been disclosed in the literature:J. Med. Chem, 37 (1997) pages 1689-1695; British Journal of Cancer, 77(1998) pages 745-752; Chem. Pharm. Bull, 40 (1995) pages 2387-2390;EP483502, USP 5216110 and JP 2306916.

In another aspect the present invention provides compounds of theformula (I) and pharmaceutically acceptable salts thereof with theprovisos that when X is S and:

a) R¹ is 4-methoxyphenyl, the benzene ring of the benzthiazole is notunsubstituted and is not substituted by 4-methyl, 4,6-dimethoxy,5-methoxy, 5,6-dimethoxy, 6-methoxy, 6-chloro or 7-methoxy;

b) R¹ is 3-methoxyphenyl, the benzene ring of the benzthiazole is notunsubstituted and is not substituted by 6-methoxy;

c) R¹ is 3,4-dimethoxyphenyl, the benzene ring of the benzthiazole isnot substituted by 6-methoxy, 4,6-dimethoxy or 5,6-dimethoxy;

d) R¹ is phenyl, the benzene ring of the benzthiazole is not substitutedby 4-methoxy, 5,6-dimethoxy, 6-hydroxy or 6-methoxy;

e) R¹ is 4-hydroxyphenyl, the benzene ring of the benzthiazole is notunsubstituted and is not substituted by 4,6-dihydroxy, 5-hydroxy,5,6-dihydroxy or 6-hydroxy;

f) R¹ is 3,4-dihydroxyphenyl, the benzene ring of the benzthiazole isnot substituted by 6-hydroxy, 4,6-dihydroxy or 5,6-dihydroxy;

g) R¹ is 2-hydroxyphenyl or 3-hydroxyphenyl, the benzene ring of thebenzthiazole is not substituted by 6-hydroxy;

h) R¹ is 4-methylphenyl, the benzene ring of the benzthiazole is notunsubstituted and is not substituted by 4-, 5- or 6-fluoro, 4-, 6- or7-methoxy, 5-chloro, 4-, 5-, 6- or 7-hydroxy, 4-, 5-, 6- or 7-acetoxy or6-nitro;

i) R¹ is 3,5-di-tert-butyl-4-hydroxyphenyl, the benzene ring of thebenztriazole is not substituted by 4- or 5-hydroxy;

and when X is S, R¹ is not 4-aminophenyl, 4-amino-3-methylphenyl or4-amino-3-halophenyl and when X is S or O, R¹ is not 4-chloro- or4-fluorophenyl when the benzene ring of the benzthiazole is substitutedby 5-hydroxy or 5-mercapto.

Particular embodiments, particular aspects and preferred features of thecompounds of this invention are as described above for the compounds foruse in treating disease conditions related to the β-estrogen receptor.

Particularly useful compounds have any of the above embodiments and alsosatisfy the equation:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>100,wherein

K_(iαA) is the K_(i) value for the agonist in ER-α;

K_(iβA) is the K_(i) value for the agonist in ER-β;

K_(iαE) is the K_(i) value for estrogen in ER-α; and

K_(iβE) is the K_(i) value for estrogen in ER-β.

Another aspect of the invention is the use of any of the above compoundembodiments for the manufacture of a medicament for the treatment orprophylaxis of Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritis orprostate cancer.

Another aspect of the invention is the use of any of the above compoundembodiments in the treatment or prophylaxis of Alzheimer's disease,anxiety disorders, depressive disorders (including post-partum andpost-menopausal depression), osteoporosis, cardiovascular disease,rheumatoid arthritis or prostate cancer.

C_(Y-Z)alkyl, unless otherwise specified, means an alkyl chaincontaining a minimum Y total carbon atoms and a maximum Z total carbonatoms. These alkyl chains may be branched or unbranched, cyclic, acyclicor a combination of cyclic and acyclic. It also includes saturated andunsaturated alkyl such as ethynyl and propenyl. For example, thefollowing substituents would be included in the general description“C₄₋₇alkyl”:

The term “oxo” means a double bonded oxygen (═O).

The compounds of the invention may contain heterocyclic substituentsthat are 5- or 6-membered ring heterocycles containing 1, 2 or 3heteroatoms each independently selected from O, N and S and additionallyhaving 0 or 1 oxo groups and 0 or 1 fused benzo rings. A nonexclusivelist containing specific examples of such heterocycles are as follows:

wherein the crossed bond represents that the heterocycle may be attachedat any available position on either the heterocycle or the benzo ring.

Some of the compounds of the present invention are capable of formingsalts with various inorganic and organic acids and bases and such saltsare also within the scope of this invention. Examples of such acidaddition salts include acetate, adipate, ascorbate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,citrate, cyclohexyl sulfamate, ethanesulfonate, fumarate, glutamate,glycolate, hemisulfate, 2-hydroxyethyl-sulfonate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, hydroxy-maleate, lactate,malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nitrate,oxalate, pamoate, persulfate, phenylacetate, phosphate, picrate,pivalate, propionate, quinate, salicylate, stearate, succinate,sulfamate, sulfanilate, sulfate, tartrate, tosylate(p-toluene-sulfonate), and undecanoate. Base salts include ammoniumsalts, alkali metal salts such as sodium, lithium and potassium salts,alkaline earth metal salts such as aluminum, calcium and magnesiumsalts, salts with organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, omithine, and so forth. Also, basic nitrogen-containing groupsmay be quaternized with such agents as: lower alkyl halides, such asmethyl, ethyl, propyl, and butyl halides; dialkyl sulfates likedimethyl, diethyl, dibutyl; diamyl sulfates; long chain halides such asdecyl, lauryl, myristyl and stearyl halides; aralkyl halides like benzylbromide and others. Non-toxic physiologically-acceptable salts arepreferred, although other salts are also useful, such as in isolating orpurifying the product.

The salts may be formed by conventional means, such as by reacting thefree base form of the product with one or more equivalents of theappropriate acid in a solvent or medium in which the salt is insoluble,or in a solvent such as water, which is removed in vacuo or by freezedrying or by exchanging the anions of an existing salt for another anionon a suitable ion-exchange resin.

Estrogen Receptor Binding Measurements

Abbreviated Procedure for Fluorescence Polarization Estrogen Receptor(ERFP) Binding Assay

A homogeneous mix-and-measure estrogen receptor (ER) binding assay whichutilizes fluorescence polarization (FP) technology is used to identifycompounds with affinity for the estrogen receptor. Purchased fromPanVera (Madison, Wis.), assay reagents include purified humanrecombinant ERα, human recombinant ERβ, ES2 screening buffer (100 mMpotassium phosphate, pH 7.4, 100 μg/mL bovine gamma globulin), andFluormone™ ES2. Fluormone™ ES2, whose formulation is proprietary toPanVera, is a fluorescein-tagged, estrogen-like molecule which exhibitsapproximately equal affinity for ERα and ERβ.

For competition binding experiments, dilutions of test compounds areprepared at 2× the final assay concentration in 0.2% DMSO in ES2Screening buffer on TECAN Genosys, and 25 μL compound/well is dispensedinto black Costar ½ volume 96-well plates. Dependent upon a lot specificK_(d) determination, 10-40 nM ERα or 1040 nM ERβ and 1 nM Fluormone ES2are then added to these plates in a final assay volume of 50 μL/well.Plates are gently shaken for at least 5 minutes to mix and incubated forat least 1 hr 45 minutes to achieve equilibrium. (Reaction mixtures arestable for up to 5 hours). After centrifugation to remove air bubbles,plates are read on an LJL Analyst or Acquest equipped with Criterionsoftware at the following settings: Fluorescence Polarization Mode;Static Polarizer on Excitation Side; Dynamic Polarizer on Emission Side;Excitation λ=485+/−10 nm; Emission λ=520+/−12.5 nm.

Polarized fluorescence intensity values are collected and subsequentlyconverted electronically to millipolarization (mp) values. Followingdata reduction and normalization with Excel and/or Prism software, %Ctrl values at the various test concentrations are used to obtain IC₅₀values via non-linear regression analysis of a four-parameter logisticequation.

Because ligand depletion is a consideration in this assay (˜40-60% inputES2 is bound in the assay), IC₅₀ values are converted to K_(i) valuesthrough application of the Kenakin formula, as outlined in the referencebelow, rather than via the more routinely-used Cheng-Prusoff formula.

Reference: Bolger et al., Rapid Screening of Environmental Chemicals forEstrogen Receptor Binding Capacity, Environmental Health Pespectives:106 (1998), 1-7.

Cell-based Assay for ER Transcriptional Activity:

ERs are ligand-dependent transcription factors that bind the promoterregions of genes at a consensus DNA sequence called the estrogenresponsive element (ERE). The ER agonist or antagonist activity of adrug was determined by measuring the amount of reporter enzyme activityexpressed from a plasmid under the control of an estrogen-responsiveelement when cells transiently transfected with ER and the reporterplasmid were exposed to drug. These experiments were conducted accordingto the following methods.

Plasmids:

Estrogen Receptors alpha (αER, Gen Bank accession #M12674), and beta(βER, Gen Bank # X99101 were cloned into the expression vector pSG5(Stratagene) and pcDNA3.1. A trimer of the vitellogenin-gene estrogenresponse element (vitERE) was synthesized as an oligonucleotide andattached to a beta-globin basal promoter in a construct named pERE3gal.This response element and promoter were removed from pERE3gal bydigestion with the endonucleases SpeI (filled with Klenow fragment) andHindIII. This blunt/Hind III fragment was cloned into theβ-galactosidase (β-gal) enhancer reporter plasmid (pBGALenh,Stratagene). αER and βER plasmids were purified using a the Endo FreeMaxi Kit (Qiagen), and the DNA concentration and purity (A260/280 ratio)were determined spectrophotometrically (Pharmacia). Only DNA withA260/280 ratio of 1.8 and a concentration of >1 ug/uL was used fortransfections.

Vitellogenin Response Element Sequence: (SEQ ID NO: 1) CTAGT CTCGAGAGGTCACTGTGACCT

AGGTCACTGTGACCTAGATCTAG GTCACTGTGACCTAC          =Spel overhang         =Xhol site          =Aflll overhang          =ERE consensus         =spacer Bgl IICells:

All Transfections are performed in 293 cells (Human Embryonic Kidneycells ATCC # CRL-1573). Cells are grown in DMEM supplemented with 10%FBS, glutamine, sodium pyruvate and penicillin/streptomycin. Cells aregrown to 80% confluency and split 1:10 or 1:20.

Transfection:

1. 293T cells are split the night before onto collagen 1150 mm plates(Biocoat Becton Dickinson #354551) at 5 million cells per plate inphenol red-free DMEM (Mediatech 17-205-CV) 10% FBS charcoal stripped(biocell #6201-31) with supplements.

2. The next day the media is changed, 1 hour prior to transfection, tofresh phenol red-free DMEM 10% FBS (charcoal stripped) and supplements.

3. Transfections are performed using the Profection Kit from Promega #El200, this kit is based on calcium phosphate mediated transfection.Reagents are added in sterile polystyrene tubes in the following order:

Solution A

20 ug ER alpha or beta (in pcDNA3.1)

50 ug Reporter (pERE3 betaGal)

1.5 ML Sterile Water

186 uL CaCl₂

* Mix gently

Solution B

1.5 ml 2×HBSS

4. Using a vortex set on low, add solution A to solution B dropwise. Theresulting solution should become milky in color. It is important to getthorough mixing at this point. Let solution stand 30 min. Vortex beforeadding to cells.

5. Add the mixture to 150-mm plates dropwise. Mix well by rocking platesback and forth and side to side gently. View cells under 20×magnification, a very fine precipitate should be seen floating on andabove cells after an hour. If you do not observe this the transfectionwill not work well. Incubate 18-20 hours.

Receptor Stimulation:

6. The day after transfection, cells are washed 2× with PBS Ca MgfreePBS containing 1 mM EGTA pH=7.6. Cells are trypsinized for 5 minwith 4 ml of trypsin (0.25%)-EDTA. Trypsin is neutralized with 6 ml DMEM(no phenol red)+10% charcoal stripped FBS. Cells are pelleted at 1000×gfor 5 min. Cell pellet is resuspended in 10 ml DMEM (no phenol red)+2%charcoal stripped FBS supplemented with glutamine and Penn/Strep and thecells are counted. Additional medium is added to dilute the cell densityto 500,000 cells/ml.

7. Cells are plated into 96 well dish (Biocoat BD #354407) at 50 ul ofcells per well (=25,000 cells/well), using a multichannel pipettor.Plates are incubated for approx. 2-4 hours to allow cells to attach.

8. Compounds are prepared at concentration of 4 mM in 100% DMSO, thendiluted into medium with supplements but no serum. The first 2 dilutionsare done in medium with no DMSO, then the remaining dilutions are inmedium plus 0.5% DMSO to keep the vehicle constant. Max controls are 10nM beta-estradiol and background controls are 0.5% DMSO. Compounds arenormally tested in the range of 10 uM to 1 nM and are prepared at twicethe concentration to be tested. The compounds are added to the cellplates, 50 ul per well. All compounds are tested with an n=4 wells forsingle poke and n=2 for 9-pt curves.

9. Cells are incubated overnight at 37° C. with the compounds.

Reporter Assay:

1. After 18-24 hr of stimulation, 100 ul of 7% CPRG cocktail is added toeach well, the plate is incubated at 37C for approximately 30 minutes to2 hours or until the OD reaches between 1.0 and 2.0. The CPRG (Roche0884308) will turn bright red as Beta Gal cleaves it.

2. The plates are read on a spectrophotometric plate reader (Spectramax,Molecular Devices) at 570 nm and raw absorbances are obtained.

Data is compiled and interpreted with Excel using XLFit or GraphPadPrism to fit concentration-response curves. The EC50 is defined as theconcentration at which 50% of the fitted maximum for a compound has beenreached.

10× Z Buffer

Sodium Phosphate (dibasic) 1.7 g 600 mM Sodium Phosphate (monobasic)0.96 g 400 mM Potassium Chloride 149 mg 100 mM Magnesium Sulfate 0.2 mLof 1 molar stock 100 mM BME 0.78 mL 500 mM Bring Final Volume to 20 mLwith De-Ionized Water

7% CPRG Cocktail

For 50 mLs:

add 3.5 mL of 50 ml of CPRG

add 3.5 mL of 10× Z Buffer

add 1 mL of 10% SDS

bring to 50 mL with DI water

Typical Results:

Absorbance values illustrating typical concentration-response curvesobtained for the ER agonist 17-β-estradiol (E) and the ER antagonistICI182,780 (A) are plotted for cells transfected with either αER or βER(see FIGS. 1 and 2).

Administration and Use

Compounds of the present invention are shown to have high selectivityfor ER-β over ER-α, and may possess agonist activity on ER-β withoutundesired uterine effects. Thus, these compounds, and compositionscontaining them, may be used as therapeutic agents in the treatment ofvarious CNS diseases related to ER-β, such as, for example, Alzheimer'sdisease.

The present invention also provides compositions comprising an effectiveamount of compounds of the present invention, including the nontoxicaddition salts, amides and esters thereof, which may, serve to providethe above-recited therapeutic benefits. Such compositions may also beprovided together with physiologically-tolerable liquid, gel or soliddiluents, adjuvants and excipients. The compounds of the presentinvention may also be combined with other compounds known to be used astherapeutic agents for the above or other indications.

These compounds and compositions may be administered by qualified healthcare professionals to humans in a manner similar to other therapeuticagents and, additionally, to other mammals for veterinary use, such aswith domestic animals. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation may also be emulsified. The activeingredient is often mixed with diluents or excipients which arephysiologically tolerable and compatible with the active ingredient.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing orpH-buffering agents, and the like.

The compositions are conventionally administered parenterally, byinjection, for example, either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient. Oral formulations include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained-release formulations,or powders.

In addition to the compounds of the present invention that display ER-βactivity, compounds of the present invention can also be employed asintermediates in the synthesis of such useful compounds.

Synthesis

Compounds within the scope of the present invention may be synthesizedchemically by means well known in the art. The following Examples aremeant to show general synthetic schemes, which may be used to producemany different variations by employing various commercially availablestarting materials. These Examples are meant only as guides on how tomake some compounds within the scope of the invention, and should not beinterpreted as limiting the scope of the invention.

In another aspect, the present invention provides a process forpreparing the compounds of the formula (I) and pharmaceuticallyacceptable salts thereof, which comprises:

a) cyclising a compound of the formula:

wherein X, R¹, R³-R⁶ are as defined hereinabove and L is hydrogen or aleaving group; orb) for preparing compounds wherein X is O, cyclising a compound of theformula:

wherein R¹, R³-R⁶ are as defined hereinabove or:c) cyclising a compound of the formula:

wherein X, R¹, R³-R⁶ are as defined hereinabove and L is hydrogen or aleaving group; ord) for preparing compounds wherein X is S, cyclising a compound of theformula:

wherein R¹, R³-R⁶ are as defined hereinabove;and thereafter, if necessary:

i) forming a pharmaceutically acceptable salt;

ii) converting a compound of the formula (I) into another compound ofthe formula (I).

EXAMPLES

Synthetic HPLC MS Example Structure Method (min) (MH⁺) 1

A, B, C 1.99 262 2

A, B 276 3

B, C 242 4

B 256 5

244 6

B 228 7

B, C 226 (M−H⁻) 8

B 242 9

D, E, F, G, H, I 2.32 296 10

F, G, H, I, J 2.29 308 11

F, G, H, I, J 2.18 321 12

F, G, H, I, J 2.30 341 13

F, G, H, I, J, K 2.26 354 14

D, E, G, H, I 2.07 262 15

G, H, I 2.03 242 16

Ref. 1, F, G, H, I 2.25 256 17

Ref. 1, F, G, H, I 2.36 276 18

Ref. 1, D, E, F, G, H, I 2.02 276 19

D, E, F, G, H, I 2.17 278 20

F, G, H, I 1.82 258 21

J, F, G, H, K, L, I 2.89 290 22

J, F, G, H, M, I 253 2.06 23

J, F, G, H, I, N 2.04 271 24

D, E, J, F, G, H, I, 2.30 341 25

J, F, G, H, I 2.32 322 26

D, E, J, F, G, H, K, I 2.24 388 27

J, F, G, H, K, I 2.22 368 28

D, E, J, F, G, H, I, M 2.05 287 29

J, F, G, H, K, I, M 2.02 267 30

D, E, J, F, G, H, I, O 1.92 278 31

P, F, G, H, I 2.18 308 32

Q, R, I 1.20 237 33

C 2.44 290.3, 292.3 34

J, F, Q, R, I 2.69 359 35

B 1.80 252.4 36

1.84 352.2, 354 (90%) (MH⁺+CH₃CN); 311.2, 313 (20%) (MH⁺)

Biological Data for Representative Compounds of this Invention: ExampleFP β-ER K_(i) (nM) FP α-ER K_(i) (nM) FP Selectivity 1 1.7 18 10 3 4.8121 25 5 290 1000 3 7 5.8 82 14 8 148 477 3 10 0.38 5.6 15 12 1.8 54 3022 1.2 14 12 23 646 2200 3

ERE ERE β-ER ERE β-ER ERE α-ER ERE α-ER Selec- Example EC₅₀ (nM) MaxEC₅₀ (nM) Max tivity 1 1.2 98 61 103 52 3 23 89 497 102 22 5 1000 981000 102 1.8 7 1.4 95 25 116 18 8 10 0.017 103 6.0 109 363 12 0.5 81 7637 153 22 0.616 98 6.4 83 10 23 298 86 1000 78 14Chemical SynthesesThe HPLC conditions used are the following unless stated otherwise: HPLC2.1×50 mm C₈ 5 μm Zorbax Stablebond column; flow rate 1.4 mL/min, lineargradient from 15% B to 90% B over 4.0 min; A=water, 0.05% TFA; B=90%acetonitrile, 10% water, 0.05% TFA, UV detection at 254 nm or DAD andpositive ionization mass spectrometry detection.

TFA: trifluoroacetic acid

DMSO: dimethylsulfoxide

DEAD: diethyl axodicarboxylate

PPh₃: triphenylphosphine

EDTA: ethylenediaminetetraacetic acid

BBr₃: boron tribromide

Example 1 4-Chloro-6-hydroxy-2-(4-hydroxyphenyl)benzoxazole

1) Synthetic method A: Synthesis of 2-amino-3-chloro-5-methoxyphenol

A solution of 3-chloro-5-methoxy-2-nitrophenol [1] (450 mg) and tin(II)chloride dihydrate (2 g, 4 eq.) in ethyl acetate (30 mL) was heatedunder reflux for 4 h. The mixture was cooled, diluted with ethyl acetateand aqueous potassium fluoride. The mixture was filtered through celite.The organic layer was washed with brine and dried over MgSO₄.Evaporation of the solvent afforded the title compound (280 mg) as apale solid. NMR (DMSO-d₆): 9.74 (m, 1H), 6.33 (d, 1H, J=2.4 Hz), 6.30(d, 1H, J=2.4 Hz), 4.19 (m, 2H), 3.60 (s, 3H).

Reference 1: Hodgson, Wignall, J. Chem. Soc., 1928, 330. Prepared from1-chloro-3,5-dimethoxybenzene by nitration with concentrated nitric acidin acetic anhydride below 10° C. to give1-chloro-3,5-dimethoxy-2-nitrobenzene, and subsequent reaction withboron tribromide (1 eq.) in dichloromethane from −78° C. to 0° C.

2) Synthetic method B: Synthesis of4-chloro-2-(4-hydroxyphenyl)-6-methoxybenzoxazole

Example 2

A solution of 2-amino-3-chloro-5-methoxyphenol (270 mg) and ethyl4-hydroxybenzimidate hydrochloride (376 mg, 1.2 eq.) in absolute ethanol(5 mL) was heated under reflux for 4 h. The mixture was cooled,partitioned between ethyl acetate and water. The organic layer was driedover MgSO₄. After evaporation of the solvent, the residue was trituratedwith methanol to give the title compound (130 mg) as a light orangesolid. NMR (DMSO-d₆): 10.35 (s, 1H), 8.00 (d, 2H, J=8.7 Hz), 7.39 (d,1H, J=2.1 Hz), 7.10 (d, 1H, J=2.1 Hz), 6.96 (d, 2H, J=8.7 Hz), 3.85 (s,3H); MS: 276 (MH⁺).

3) Synthetic method C: Synthesis of4-chloro-6-hydroxy-2-(4-hydroxyphenyl)benzoxazole

To a suspension of 4-chloro-2-(4-hydroxyphenyl)-6-methoxybenzoxazole(240 mg) in dichloromethane (5 mL) cooled at −78° C. was added borontribromide (5 mL, 1M solution in dichloromethane, 5.7 eq.). The mixturewas stirred at −78° C. for 10 min, warmed to room temperature andstirred for 3 h. The mixture was poured onto ice/water and extractedwith ethyl acetate. The organic layer was washed with brine and driedover MgSO₄. Chromatography on a silica gel column (eluant: acetone-dichloromethane, gradient from 0:100 to 5:95) and trituration of theresulting solid in dichloromethane afforded the title compound (53 mg)as a solid. NMR (DMSO-d₆): 10.31 (s, 1H), 10.17 (s, 1H), 7.98 (d, 2H,J=8.4 Hz), 7.06 (s, 1H), 6.95 (d, 2H, J=8.4 Hz), 6.87 (s, 1H); MS: 262(MH⁺); HPLC t_(R): 1.99 min.

Example 3 6-Hydroxy-2-(4-hydroxyphenyl)-4-methylbenzoxazole

1) Synthesis of 2-(4-hydroxyphenyl)-6-methoxy-4-methylbenzoxazole(Example 4)

According to synthetic method B, from 2-amino-5-methoxy-3-methylphenol[2] (440 mg) was obtained the title compound (340 mg) as a light orangesolid. MS: 256 (MH⁺).

Reference 2: Musso H; Beecken H, Chem. Ber. 1961, 94, 585; made from3,5-dimethoxytoluene by nitration and monodeprotection of the 3-methoxywith BBr₃ similarly to Ref. 1 followed by reduction of the nitro groupto the amino by hydrogenation with palladium on charcoal.

2) According to synthetic method C, the above compound (220 mg) wasconverted to 6-hydroxy-2-(4-hydroxyphenyl)-4-methylbenzoxazole (112 mg)as a light pinkish powder. NMR (DMSO-d₆): 10.14 (s, 1H), 9.58 (s, 1H),7.94 (d, 2H, J=8.7 Hz), 6.92 (d, 2H, J=8.7 Hz), 6.84 (s, 1H), 6.63 (s,1H), 2.46 (s, 3H); MS: 242 (MH⁺).

Example 5 4,6-Dihydroxy-2-(4-hydroxyphenyl)benzoxazole

A mixture of 1-nitro-2,4,6-trihydroxybenzene (1 g) and 10% palladium oncharcoal (200 mg) in absolute ethanol (10 mL) was stirred for 18 h atroom temperature under a 50-PSI atmosphere of hydrogen. The solids werefiltered rapidly. To the resulting filtrate was added ethyl4-hydroxybenzimidate hydrochloride (1.17 g). The mixture was heated atreflux for 5 h under nitrogen, cooled and partitioned between ethylacetate and water. The organic layer was washed with water and brine,and dried over MgSO4. Chromatography on silica gel (eluant:acetone-dichloromethane, gradient 10:90 to 20:80) and trituration of theresulting solid with ether afforded the title compound (28 mg) as alight pinkish solid. NMR (DMSO-d₆): 10.15 (m, 2H), 9.53 (s, 1H), 7.91(d, 2H, J=8.7 Hz), 6.92 (d, 2H, J=8.7 Hz), 6.48 (d, 1H, J=1.5 Hz), 6.26(d, 1H, J=1.5 Hz); MS: 244 (MH⁺).

Example 6 6-Hydroxy-2-(4-hydroxyphenyl)benzoxazole

According to synthetic method B except that pyridine (300 μL) was added,from 4-aminoresorcinol hydrochloride (435 mg) and ethyl4-hydroxybenzimidate hydrochloride was obtained the title compound (432mg) as an off-white solid. The work-up of the reaction was modified asfollows: After completion of the reaction, the mixture was diluted withether and water, and filtered. The solids were washed with water andether, and dried under high vacuum. NMR (DMSO-d₆): 10.23 (s br, 1H),9.76 (s br, 1H), 7.95 (d, 2H, J=8.4 Hz), 7.50 (d, 1H, J=8.4 Hz), 7.05(d, 1H, J=1.8 Hz), 6.94 (d, 2H, J=8.4 Hz), 6.81 (dd, 1H, J=8.4 Hz,J′=1.8 Hz); MS: 228 (MH⁺).

Example 7 5-Hydroxy-2-(4-hydroxyphenyl)benzoxazole

According to synthetic method B, from 2-amino-4-methoxyphenol [3] (500mg) and ethyl 4-hydroxybenzimidate hydrochloride was obtained2-(4-hydroxyphenyl)-5-methoxybenzoxazole (676 mg; Example 8). NMR(DMSO-d₆): 10.28 (s, 1H), 8.01 (d, 2H, J=8.7 Hz), 7.61 (d, 1H, J=9 Hz),7.28 (d, 1H, J=2.4 Hz), 6.94 (m, 3H), 3.82 (s, 3H); MS: 242 (MH⁺). Thiscompound (452 mg) was converted to the title compound (121 mg) accordingto synthetic method C. NMR (DMSO-d₆): 10.24 (s, 1H), 9.42 (s br, 1H),7.99 (d, 2H, J=8.7 Hz), 7.49 (d, 1H, J=9 Hz), 7.02 (d, 1H, J=2.1 Hz),6.94 (d, 2H, J=8.7 Hz), 6.78 (dd, 1H, J=9 Hz, J′=2.1 Hz); MS: 226(M−H⁻).Reference 3: Lok R, Leone R E, Williams A J, J. Org. Chem. 1996, 61,3289.

Example 9 4-Chloro-2-(2-chloro-4-hydroxy-phenyl)-benzooxazol-6-ol

Synthetic method D: Synthesis of 2-chloro-4-methoxy-benzoic acid methylester

To 2-chloro-4-hydroxy-benzoic acid hydrate (1.1 g) in DMF (20 mL) wasadded K₂CO₃ (2.4 g) and methyl iodide (0.75 mL). After 2 h, water wasadded to the reaction mixture and extracted with ethyl acetate. Theorganic layer was washed with brine (3×) and dried over MgSO₄. Flashchromatography on silica gel eluting with 10% ethyl acetate-hexaneafforded 1.13 g (95%) of the title compound as an oil. MS: 201 (MH⁺),HPLC t_(R): 2.52 min.

Synthetic method E: Synthesis of 2-chloro-4-methoxy-benzoic acid

To 2-chloro-4-methoxy-benzoic acid methyl ester (1.1 g) inTHF/MeOH/water (12 mL/3 mL/3 mL) at room temperature was added LiOH (461mg) dissolved in water. After 2 h, the reaction mixture was adjusted topH 4 with 1N HCl and partitioned between ethyl acetate and water. Theorganic layer was washed with brine and dried over MgSO₄. Trituration ofthe resulting solid in ether afforded 1.0 g (98%) of the title compound.MS: 187 (MH⁺), HPLC t_(R): 2.04 min.

Synthetic method F: Synthesis of 2-amino-3-chloro-5-methoxyphenol

To 3-chloro-5-methoxy-2-nitrophenol, synthetic method A [1], (200 mg) in95% EtOH (10 mL) was added 5% Ru/C (20 mg) and hydrazine (0.36 mL). Themixture was placed in an oil bath and heated to 85° C. for 2 h. Afterreaction cooled, the mixture was diluted with ethyl acetate and filteredthrough a pad of celite and concentrated. Flash chromatography on silicagel eluting with 30% ethyl acetate-hexane afforded 138 mg (76%) of thetitle compound as a solid. MS: 174 (MH⁺), HPLC t_(R): 0.84 min.

Synthetic method G: Synthesis of2-chloro-N-(2-chloro-6-hydroxy-4-methoxy-phenyl)-4-methoxy-benzamide.

To 2-chloro-4-methoxy-benzoic acid (100 mg) in CH₂Cl₂ (5 mL) was addedoxalyl chloride (0.05 mL) and 2 drops of DMF. The reaction mixture wasallowed to stir for 2 h and then concentrated to dryness. The resultingacid chloride was taken up in CH₂Cl₂ and added drop wise to a coolmixture of 2-amino-3-chloro-5-methoxyphenol (94 mg), 10% Na₂CO₃ (2.5mL), and CH₂Cl₂ (5 mL) placed in an ice water bath. After 2 h, water wasadded to the reaction and extracted with additional CH₂Cl₂ and theorganic layer was dried over Na₂SO₄. Flash chromatography on silica geleluting with 0% to 30% ethyl acetate-hexane afforded 100 mg (54%) of thetitle compound as an solid. MS: 342 (MH⁺), HPLC t_(R): 2.54 min.

Synthetic method H: Synthesis of4-chloro-2-(2-chloro-4-methoxy-phenyl)-6-methoxy-benzooxazole

Reference 4. Wang, F.; Hauske, J. R.; Tetrahedron Lett. 1997, 38 (37)6529-6532

2-Chloro-N-(2-chloro-6-hydroxy-4-methoxy-phenyl)-4-methoxy-benzamide (40mg) was completely dissolved in THF (5 mL) and PPH₃ (46 mg) was addedand the mixture was stirred until all PPH₃ dissolved. To this mixturewas added drop wise DEAD (0.03 mL) diluted in THF (0.5 mL). The mixturewas stirred at room temperature for 2 h and the reaction mixture wasdiluted with ethyl acetate and washed with water, brine, dried overMgSO₄ and concentrated. Flash chromatography on silica gel eluting with10% to 30% ethyl acetate-hexane afforded 32 mg (85%) of the titlecompound as a solid. MS: 324 (MH⁺), HPLC t_(R): 3.18 min.

Synthetic method I: Synthesis of4-chloro-2-(2-chloro-4-hydroxy-phenyl)-benzooxazol-6-ol

To 4-chloro-2-(2-chloro-4-methoxy-phenyl)-6-methoxy-benzooxazole (52 mg)in CH₂Cl₂ (2 mL) placed in and ice water bath was added drop wise 1.0 MBBr₃ in CH₂Cl₂ (0.96 mL, 6 eq). After the reaction stirred over night,the mixture was placed in an ice water bath and excess MeOH was addeddrop wise to quench excess BBr₃ and the mixture was stirred for anadditional 20 min and concentrated. Flash chromatography on silica geleluting with 0% to 40% ethyl acetate-hexane afforded 35 mg (74%) of the.title compound as a solid. MS: 296 (MH⁺), HPLC t_(R): 2.32 min.

Example 10 7-Bromo-2-(4-hydroxy-phenyl)-benzooxazol-5-ol

Synthetic method J: Synthesis of 2-bromo-4-methoxy-6-nitro-phenol

4-Methoxy-2-nitro-phenol (10 g) was dissolved in glacial acetic acid (60mL) and CH₃CO₂Na (8.2 g) was added. Next, bromine (3 mL) dissolved inglacial acetic acid (12 mL) was added drop wise to the stirring solutionat room temperature. After complete addition of bromine, the mixture wasstirred for 30 min at room temperature and then placed in an oil bath at75° C. for 2 h. After reaction mixture cooled to room temperature,concentrated HCl (500 mL) was slowly added to the mixture followed byaddition of ethyl acetate (500 mL). The layers were separated and theorganic layer was washed with water, brine, dried (Na₂SO₄). Flashchromatography on silica gel eluting with 5% ethyl acetate-hexaneafforded 8.8 g (60%) of the title compound as a solid. MS: 218 (MH⁺−30),HPLC t_(R): 2.40 min.

According to synthetic methods F, G, H (except the reaction mixture washeated in an oil bath at 85° C. for 2 h), and I was obtained7-bromo-2-(4-hydroxy-phenyl)-benzooxazol-5-ol. MS: 308 (MH⁺), HPLCt_(R): 2.29 min. NMR (DMSO-d₆): 10.34 (s, 1H), 9.82 (s, 1H), 7.99 (d,2H, J=8.4 Hz), 6.95-7.04 (m, 4H).

Example 12 7-Bromo-2-(3-chloro-4-hydroxy-phenyl)-benzooxazol-5-ol

According to synthetic methods J (using 4-methoxy-2-nitro-phenol), F, G(using 3-chloro-4-methoxy-benzoic acid), H (except the reaction mixturewas heated in an oil bath at 85° C. for 2 h), and I the title compoundwas obtained. MS: 341 (MH⁺), HPLC t_(R): 2.30 min. NMR (DMSO-d₆): 11.17(s, 1H), 9.85 (s, 1H), 8.04 (s, 1H), 7.95 (d, 1H, J=8.2 Hz), 7.18 (d,1H, J=8.3 Hz), 7.05 (s, 1H), 7.02 (s, 1H).

Example 13 2-(4-Hydroxy-phenyl)-7-iodo-benzooxazol-5-ol

Synthetic method K: Synthesis of7-iodo-5-methoxy-2-(4-methoxy-phenyl)-benzooxazole

To 7-bromo-5-methoxy-2-(4-methoxy-phenyl)-benzooxazole (100 mg, fromexample 10, synthetic methods F, G, H, J), was added CuI (285 mg), KI(497 mg) and DMSO (5 mL). The mixture was placed in an oil bath andheated to 180° C. for 4 h. The mixture was cooled to room temperatureand diluted with ethyl acetate and washed with brine (3×), dried(MgSO₄), and concentrated. The crude solid was taken up in ethyl acetateand filtered through a pad of celite and the filtrate was concentratedto dryness. Flash chromatography on silica gel eluting with 20% ethylacetate-hexane afforded 80 mg (70%) of the title compound as a solid.MS: 382 (MH⁺), HPLC t_(R): 3.00 min. According to synthetic method I wasobtained 2-(4-Hydroxy-phenyl)-7-iodo-benzooxazol-5-ol. MS: 354 (MH⁺),HPLC t_(R): 2.26 min.

Example 21 7-Chloro-2-(4-hydroxy-phenyl)-benzooxazol-5-ol

Synthetic method L: Synthesis of7-chloro-5-methoxy-2-(4-methoxy-phenyl)-benzooxazole

To 7-iodo-5-methoxy-2-(4-methoxy-phenyl)-benzooxazole (150 mg) in DMF (6mL) was added CuCl (195 mg). The mixture was placed in an oil bath andheated to 150° C. for 3 h. The mixture was cooled to room temperatureand diluted with ethyl acetate and washed with 1N HCl, brine (3×), dried(MgSO₄), and concentrated. Flash chromatography on silica gel elutingwith 20% ethyl acetate-hexane afforded 100 mg (88%) of the titlecompound as a solid. MS: 290 (MH⁺), HPLC t_(R): 2.89 min.

According to synthetic method I was obtained7-chloro-2-(4-hydroxy-phenyl)-benzooxazol-5-ol. MS: 262 (MH⁺), HPLCt_(R): 2.09 min.

Example 22 5-Hydroxy-2-(4-hydroxy-phenyl)-benzooxazole-7-carbonitrile

Synthetic method M: Synthesis of5-hydroxy-2-(4-hydroxy-phenyl)-benzooxazole-7-carbonitrile

To 7-bromo-2-(4-hydroxy-phenyl)-benzooxazol-5-ol (96 mg) in DMF (3 mL)was added CuCN (84 mg). The mixture was placed in an oil bath and heatedto 150° C. for 3 h. The mixture was cooled to room temperature anddiluted with ethyl acetate and washed with 1N HCl, saturated aqueousEDTA, brine (3×), dried (Na₂SO₄), and concentrated. Flash chromatographyon silica gel eluting with 20% ethyl acetate-hexane afforded 20 mg (25%)of the title compound as a solid. MS: 253 (MH⁺), HPLC t_(R): 2.06 min.

Example 23 5-Hydroxy-2-(4-hydroxy-phenyl)-benzooxazole-7-carboxylic acidamide

Synthetic method N: Synthesis of5-methoxy-2-(4-methoxy-phenyl)-benzooxazole-7-carbonitrile

To 7-bromo-5-methoxy-2-(4-methoxy-phenyl)-benzooxazole (200 mg) in DMF(5 mL) was added CuCN (80 mg). The mixture was placed in an oil bath andheated to 150° C. for 3 h. The mixture was cooled to room temperatureand diluted with ethyl acetate and washed with 1N HCl, brine (3×), dried(Na₂SO₄), and concentrated. Flash chromatography on silica gel elutingwith 20% ethyl acetate-hexane afforded 85 mg (50%) of the title compoundas a solid. MS: 281 (MH⁺), HPLC t_(R): 2.71 min.

According to synthetic method I was obtained5-hydroxy-2-(4-hydroxy-phenyl)-benzooxazole-7-carboxylic acid amide byresidual acid hydrolysis of the nitrile substituent upon concentrationof the crude reaction mixture. MS: 271 (MH⁺), HPLC t_(R): 2.04 min.

Example 302-(2-Cyano-4-hydroxy-phenyl)-5-hydroxy-benzooxazole-7-carbonitrile

Synthetic method O: Synthesis of2-(2-cyano-4-hydroxy-phenyl)-5-hydroxy-benzooxazole-7-carbonitrile

To 2-(2-chloro-4-hydroxy-phenyl)-7-iodo-benzooxazol-5-ol (279 mg) in DMF(5 mL) was added CuCN (97 mg). The mixture was placed in an oil bath andheated to 150° C. for 3 h. The mixture was cooled to room temperatureand diluted with ethyl acetate and washed with 1N HCl, saturated aqueousEDTA, brine (3×), dried (Na₂SO₄), and concentrated. Material waspurified by preparative LC/MS. MS: 278 (MH⁺), HPLC t_(R): 1.92 min. TheHPLC conditions used are the following: HPLC Waters Corp. Novapak HR™ C18 RCM 40×100 mm 6 μm particle; flow rate 40 mL/min, linear gradientfrom 35% B to 65% B over 15 min; A=water, 0.1% TFA; B=MeOH, UV detectionat 254 nm and positive ionization mass spectrometry detection

Example 31 6-Bromo-2-(4-hydroxy-phenyl)-benzooxazol-5-ol

Synthetic method P: Synthesis of 1 -bromo-2,5-dimethoxy-4-nitro-benzene

The title compound was synthesized according to the methods describe byReference 5: Jean-Luc Grenier, Jean-Pierre Catteau and Philippe Cotelle,Synthetic Communications, 29(7), 1201-1208 (1999).

According to synthetic methods F, G, H and I, was obtained6-bromo-2-(4-hydroxy-phenyl)-benzooxazol-5-ol. MS: 308 (MH⁺), HPLCt_(R): 2.18 min.

Example 32 4-(6-Hydroxy-benzooxazol-2-yl)-thiazolidin-2-one

Reference 6. Steven W. Goldstien and Paul J. Dambek, J. HeterocyclicChem. 1990, 27, 225.

Synthetic method Q: Synthesis of 2-oxo-thiazolidine-4-carboxylic acid(2-hydroxy-4-methoxy-phenyl)-amide

To 2-oxo-thiazolidine-4-carboxylic acid (492 mg) in CH₂Cl₂ (10 mL) wasadded oxalyl chloride (0.35 mL) and 2 drops of DMF. The reaction mixturewas allowed to stir for 2 h and then concentrated to dryness. Theresulting acid chloride was taken up in CH₂Cl₂ and added drop wise to acool mixture of 2-amino-5-methoxy-phenol (490 mg), Et₃N (1.56 mL), andCH₂Cl₂ (10 mL) placed in an ice water bath. After 2 h, water and CH₂Cl₂was added to the reaction mixture and layers separated. The organiclayer was washed with 1N HCl. To the HCl aqueous layer was added brineand CH₂Cl₂ to salt-out the title compound, the organic layers werecombined, dried (Na₂SO₄) and concentrated to afford 448 mg (60%) of thetitle compound. The crude amide was used directly in the next step. MS:269 (MH⁺), HPLC t_(R): 1.37 min.

Synthetic method R: Synthesis of4-(6-methoxy-benzooxazol-2-yl)-thiazolidin-2-one

To 2-oxo-thiazolidine-4-carboxylic acid(2-hydroxy-4-methoxy-phenyl)-amide (50 mg) in p-xylene (3 mL) was addedpyridinium p-toluenesolfonate (42 mg). The reaction mixture was refluxedfor 2 h and then allowed to cool to room temperature. The mixture wasdiluted with ethyl acetate and water was added. The layers wereseparated and the organic layer was washed with brine, dried (Na₂SO₄),and concentrated. Flash chromatography on silica gel eluting with 3%methanol-methylene chloride afforded 42 mg (90%) of the title compoundas a solid. MS: 251 (MH⁺), HPLC t_(R): 1.60 min.

According to synthetic method I was obtained4-(6-hydroxy-benzooxazol-2-yl)-thiazolidin-2-one 30 mg (63%). MS: 237(MH⁺), HPLC t_(R): 1.20 min.

Example 33 7-Bromo-2-phenyl-benzooxazol-5-ol

tert-Butyl-chloro-silane (0.25 mL) was added to a mixture of2-bromo-4-methoxy-6-nitro-phenol (0.124 g), chromium (II) chloride(0.012 g) and manganese (0) powder (0.137 g) in dimethylformamide (3mL). The mixture was subjected to microwaves for 4 min at 150° C.Benzaldehyde (0.06 mL) was added and the reaction was subjected tomicrowaves for 6 min at 150° C. Water (0.5 mL) was added, then after 30min the mixture was filtered through a pad of celite. The aboveprocedure was repeated three more times. The combined filtrate waspartitioned between dilute aq. HCl (100 mL) and ethyl acetate (100 mL).The organic layer was dried over sodium sulfate, filtered through celiteand concentrated. Chromatography on silica gel (eluant: gradient 0 to30% ethyl acetate:hexane) afforded7-bromo-5-methoxy-2-phenyl-benzooxazole (0.13 g). MS: 304 (95%), 306.4(100%) (MH⁺); HPLC t_(R): 2.79 min.

Reference 7: J. Org. Chem. 2001, 66, 991-996.7-Bromo-5-methoxy-2-phenyl-benzooxazole was deprotected using syntheticmethod C to give the title compound (0.060 g) after purification onsilica gel (eluant: 5 to 30% ethyl acetate:hexane). MS: 290.3, 292.3(MH⁺); HPLC t_(R): 2.44 min.

Example 35 2-(1H-Indazol-5-yl)-benzooxazol-6-ol

1H-Indazole-5-carbonitrile hydrochloride (1.5 g) was suspended inethanol (15 mL) at 0° C. The mixture was saturated with hydrogenchloride while the temperature was raised to room temperature. Thereaction was then left overnight. Diethylether was added and theresulting precipitate, 1H-indazole-5-carboximidic acid ethyl ester.2HCl(1.39 g), was collected and dried in under high vacuum. According tosynthetic method B except that pyridine (540 ?L) was added, from4-amino-benzene-1,3-diol hydrochloride (0.36 mg) and1H-indazole-5-carboximidic acid ethyl ester hydrochloride (0.71 g) wasobtained the title compound (0.34 mg) as an off-white solid. The work-upof the reaction was modified as follows: After completion of thereaction, the mixture was diluted with ether and water, and filtered.The solids were washed with water and ether, and dried under highvacuum. MS: 252.4 (MH⁺); HPLC t_(R): 1.80 min.

Example 36 5-(7-Bromo-5-methoxy-benzooxazol-2-yl)-pyrrolidin-2-one

2-Amino-6-bromo-4-methoxy-phenol (0.40 g),1,3-dimethylaminopropyl)-3-ethylcarbodiimide (1.06 g),1-hydroxybenzotriazole (0.50 g), dimethylaminopyridine (0.22 g) andDL-5-oxo-pyrrolidine-2-carboxylic acid (0.25 g) were reacted together inmethylene chloride (7.3 mL). After 3 h, the reaction was diluted withmethylene chloride (10 mL) and washed successively with 1N HCl (10 mL),sat. aq. NaHCO₃ (10 mL), sat. aq. NaCl (10 mL). The organic layer wasdried over sodium sulfate, filtered through celite and concentrated.Chromatography on silica gel (eluant: 0 to 20% methanol:methylenechloride) afforded 5-oxo-pyrrolidine-2-carboxylic acid(3-bromo-2-hydroxy-5-methoxy-phenyl)-amide (0.18 g). MS: 370.2 (100%),372.2 (90%) (MH⁺); HPLC t_(R): 1.51 min. 5-Oxo-pyrrolidine-2-carboxylicacid (3-bromo-2-hydroxy-5-methoxy-phenyl)-amide was cyclized accordingto synthetic method R to obtain the title compound (0.09 g) afterpurification on silica gel (eluant: 0 to 30% methanol:methylenechloride). MS: 352.2 (100%), 354.2 (95%) (MH⁺+CH₃CN), 311.2 (20%), 313.2(20%) (MH⁺).; HPLC t_(R): 1.84 min.

Reference Example 37 5-Methoxy-2-aminobenzenethiol hydrochloride

Prepared following literature procedure: Can. J. Chem. 43,1965, 2610.

Example 38 6-Hydroxy-2-R-benzothiazol

5-Methoxy-2-aminobenzenethiol hydrochloride was dissolved in1-methyl-2-pyrrolidinone. After triethylamine (1 eq) was added, themixture was stirred at room temperature for 10 min. Acid chloride (1 eq)was then added and the mixture was heated at 100° C. for 1 h. Themixture was then cooled to room temperature and 1N NaOH was added to PH9. The solid was collected by filtration and washed with water. Thesolid was further dried on vacuum to give 6-methoxy-2-R-benzothiazol(yield step 1) which was treated with boron tribromide under standardprocedure to give 6-hydroxy-2-R-benzothiazol (yield step 2).    R group

yield on step 1 68 29 70 85 yield on step 2 66 68 39 85 Mass Spec MH⁺234 218 253 297

Example 39 4-Methyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

To a solution of 4-bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole(1.0 g, 2.86 mmol) in dry toluene (25 mL) was added potassium carbonate(4.2 g, 30.4 mmol), methylboronic acid (0.92 g, 15.3 mmol) andtetrakis(triphenylphosphine)palladium (0.44 g, 0.38 mmol). The mixturewas heated to 100° C. for 24 h, then cooled to room temperature. Themixture was diluted with ethylacetate and washed with water, saturatedsodium carbonate and brine. After chromatographic purification gave4-methyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole as a white solid(0.63 g, 77% yield). Mass spec: MH⁺=286.

Example 40 4-Methyl-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Methyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (174 mg, 0.61mmol) was treated with boron tribromide under standard condition to give4-methyl-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole (105 mg, 67%yield) as a yellow solid. Mass spec: MH⁺=258.

Example 41 4-Bromomethyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

4-Methyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.53 g, 1.87mmol), N-bromosuccinimide (0.33 g, 1.87mmol) and benzoyl peroxide (9 mg)were suspended in carbon tetrachloride (6 mL) and refluxed for 3.5 h,then cooled to room temperature. Solvent was evaporated and the mixturewas purified by chromatography to give4-bromomethyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.47 g, 70%yield) as a white solid. Mass spec: MH⁺=364.

Example 42 4-Cyanomethyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

To a solution of4-bromomethyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole ( 0.47 g,1.29 mmol) in ethanol (6 mL) was added a solution of potassium cyanide(0.1 g, 1.54 mmol) in water (0.5 mL). The mixture was refluxed for 1.5h, then cooled to room temperature. Ethanol was evaporated and themixture was extracted with ethylacetate. After chromatographicpurification gave4-cyanomethyl-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole as a yellowsolid (0.29 g, 73% yield). Mass spec: MH⁺=311.

Example 43 4-Cyanomethyl-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Methylcyano-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.29 g, 0.94mmol) was treated with boron tribromide under standard condition to give4cyanomethyl-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole (116 mg, 44%yield) as a yellow solid. Mass spec: MH⁺=283.

Example 444-Trimethylsilylacetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

4-Bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.3 g, 0.857mmol), (trimethylsilyl)acetylene (0.48 mL, 3.43 mmol), triethylamine(0.48 mL, 3.43 mmol) and tetrakis(triphenylphosphine)palladium (0.2 g,0.171 mmol) were suspended in THF (6 mL) in a sealed tube and heated to70° C. for 24 h, then cooled to room temperature. Ethylacetate and waterwere added, the ethylacetate layer was washed with brine. Afterchromatigraphic purification gave4-trimethylsilylacetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole(0.132 g, 42% yield) as a yellow solid. Mass spec: MH⁺=368.

Example 45 4-Acetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

To a solution of4-trimethylsilylacetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole(0.216 g, 0.588 mmol) in THF (5 mL) was added 1N sodium hydroxide (1.18mL, 1.18 mmol) and the solution was stirred at room temperature for 3 h.THF was then evaporated. Water and ethylacetate were added. Combinedethylacetate were concentrated. After chromatigraphic purification gave4-acetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.113 g, 65%yield) as a yellow solid. Mass spec: MH⁺=296

Example 46 4-Acetylene-6 hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Acetylene-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (93 mg, 0.315mmol) was treated with boron tribromide under standard condition to give4-acetylene-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole (47 mg, 46%yield) as a yellow solid. Mass spec: MH⁺=268

Example 47 4-Methylcarboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

4-Bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (1.0 g, 2.86 mmol),triethylamine (0.96 mL, 7.15 mmol) palladium acetate (31 mg, 0.143 mmol)and 1,3-bis(diphenylphosphino)propane (57 mg, 0.143 mmol) were suspendedin methanol (7 mL) and DMSO (7 mL). The mixture was heated to 75° C. andbubbled with CO for 20 min. The mixture was then heated under CO for 48h. After cooling to room temperature, brine was added. The mixture wasextracted with ethylacetate. Combined ethylacetate were concentrated.After chromatographic purification gave4-methylcarboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.37 g,39% yield) as a white solid. Mass spec: MH⁺=330.

Example 48 4-Carboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole

To a solution of4-methylcarboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.37 g,1.12 mmol) in THF (6 mL) and water (3 mL) was added 1N sodium hydroxide(2.24 mL, 2.24 mmol) and the solution was stirred at room temperaturefor 24 h. THF was then evaporated. 1 N HCl was added to PH 1. The solidwas collected by filtration and washed with water to give4-carboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (0.29 g, 82%yield) as a white solid. Mass spec: MH⁺=316.

Example 49 4-Carboxy-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Carboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (50 mg, 0.159mmol) was treated with boron tribromide under standard condition to give4-carboxy-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole (34 mg, 74%yield) as a yellow solid. Mass spec: MH⁺=288. 1H NMR (DMSO-d6): 10.24(s, 1H), 10.16 (s, 1H), 7.91 (d, 2H), 7.67 (s, 1H), 7.48 (s, 1H), 6.95(d, 2H).

Example 50 4-Carboxyamide-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Carboxy-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (1 eq),1-hydroxybenzotriazole (2.4 eq), dimethylamine or methylamine (3.7 eq)and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.3 eq)were suspended in DMF and stirred at room temperature for 5 min.Triethylamine (4.1 eq) was added and the mixture was stirred at roomtemperature for 24 h. [when R₁═R₂═H, only1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.05 eq)and 1-hydroxybenzotriazole ammonia (2.46 eq) were added]. Saturatedsodium bicarbonate was added and the mixture was extrated withethylacetate. Combined ethylacetate were washed with brine,concentrated. After chromatographic purification gave the product (yieldstep 1) which was further treated with boron tribromide under standardcondition to give4-carboxyamide-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole (yield step2). R₁/R₂ R₁═R₂═Me R₁═H, R₂═Me R₁═R₂═H yield on step 1 86 67 91 yield onstep 2 100 70 44 Mass Spec MH⁺ 315 301 287

Example 51 6-Methoxy-2-(2-bromo-3-methoxy-phenyl)-benzothiazole

5-Methoxy-2-aminobenzenethiol hydrochloride (83 g, 4.33 mmol) wasdissolved in 1-methyl-2-pyrrolidinone (10 mL). After triethylamine (0.60mL, 4.33 mmol) was added, the mixture was stirred at room temperaturefor 10 min. 2-Bromo-3-methoxybenzoyl chloride (1.08 g, 4.33 mmol) in1-methyl-2-pyrrolidinone (10 mL) was then added and the mixture washeated at 100° C. for 40 min. The mixture was cooled to room temperatureand 1N NaOH was added to PH 9. The mixture was extracted withethylacetate and combined ethylacetate were washed with brine,concentrated. After chromatographic purification gave6-methoxy-2-(2-bromo-3-methoxy-phenyl)-benzothiazole (0.98 g, 65% yield)as a yellow solid. Mass spec: MH⁺=350.

Example 52 6-Hydroxy-2-(2-bromo-3-hydroxy-phenyl)-benzothiazole

6-Methoxy-2-(2-bromo-3-methoxy-phenyl)-benzothiazole (0.144 g, 0.411mmol) was treated with boron tribromide under standard condition to give6-hydroxy-2-(2-bromo-3-hydroxy-phenyl)-benzothiazole (35 mg, 26% yield)as a yellow solid. Mass spec: MH⁺=322.

Example 53 6-Methoxy-2-(2-methyl-3-methoxy-phenyl)-benzothiazole

To a solution of 6-methoxy-2-(2-bromo-3-methoxy-phenyl)-benzothiazole(0.128, 0.366 mmol) in dry toluene (4 mL) was added potassium carbonate(0.404 g, 2.92 mmol), methylboronic acid (88 mg, 1.47 mmol) andtetrakis(triphenylphosphine)palladium (42 mg, 0.036 mmol). The mixturewas heated to 100° C. for 3 h, then cooled to room temperature. Themixture was diluted with ethylacetate and washed with water, saturatedsodium carbonate and brine. After chromatographic purification gave6-methoxy-2-(2-methyl-3-methoxy-phenyl)-benzothiazole (66 mg, 63% yield)as a white solid. Mass spec: MH⁺=286.

Example 54 6-Hydroxy-2-(2-methyl-3-hydroxy-phenyl)-benzothiazole

6-Methoxy-2-(2-methyl-3-methoxy-phenyl)-benzothiazole (64 mg, 0.224mmol) was treated with boron tribromide under standard condition to give6-hydroxy-2-(2-methyl-3-hydroxy-phenyl)-benzothiazole (52 mg, 90% yield)as a yellow solid. Mass spec: MH⁺=258.

Example 55 6-Methoxy-2-(3-methoxy-phenyl)-benzothiazole

To a solution of 6-methoxy-2-(2-bromo-3-methoxy-phenyl)-benzothiazole(0.182, 0.52 mmol) in dry DMF (3 mL) was added cesium carbonate (0.51 g,1.57 mmol), triethylborane (0.58 mL, 1M in THF, 0.58 mmol) and1,1′-bis(diphenylphosphino)ferrocene palladium dichloridedichloromethane (18 mg, 0.022 mmol). The mixture was heated to 50° C.for 24 h, then cooled to room temperature. Saturated sodium bicarbonatewas added and the mixture was extracted with ethylacetate. Combinedethylacetate were washed with brine, concentrated. After chromatographicpurification gave 6-methoxy-2-(3-methoxy-phenyl)-benzothiazole (54 mg,38% yield) as a white solid. Mass spec: MH⁺=272.

Example 56 6-Hydroxy-2-(3-hydroxy-phenyl)-benzothiazole

6-Methoxy-2-(3-methoxy-phenyl)-benzothiazole (54 mg, 0.199 mmol) wastreated with boron tribromide under standard condition to give6-hydroxy-2-(3-hydroxy-phenyl)-benzothiazole (15 mg, 31% yield) as ayellow solid. Mass spec: MH⁺=244.

Example 57 6-Hydroxy-2-(2-R-3-hydroxy-phenyl)-benzothiazole

5-Methoxy-2-aminobenzenethiol hydrochloride (1 eq) was dissolved in1-methyl-2-pyrrolidinone. After triethylamine (1 eq) was added, themixture was stirred at room temperature for 10 min. 2-R-3-methoxybenzoylchloride (1 eq) in 1-methyl-2-pyrrolidinone was then added and themixture was heated at 100° C. (reaction time in the table). The mixturewas cooled to room temperature and 1N NaOH was added to PH 9. Themixture was extracted with ethylacetate and combined ethylacetate werewashed with brine, concentrated. After chromatographic purification gave6-methoxy-2-(2-R-3-methoxy-phenyl)-benzothiazole (yield step 1) whichwas treated with boron tribromide under standard condition to give thetitle compound (yield step 2).   R group   —CH₂CH₃   —CH(CH₃)₂

Reaction time on step 1  45 min 300 min  30 min Yield on step 1  49  25 15 Yield on step 2  77 100  43 Mass spec MH⁺ 272 286 284

Example 58 4-Cyano-6-methoxy-2-(2-ethyl-3-methoxy-phenyl)-benzothiazole

The procedure was the same with4-cyano-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole except using2-ethyl-3-methoxy-benzoyl chloride instead of p-anisoyl chloride. Massspec: MH⁺=325.

Example 59 4-Cyano-6-hydroxy-2-(2-ethyl-3-hydroxy-phenyl)-benzothiazole

4-Cyano-6-methoxy-2-(2-ethyl-3-methoxy-phenyl)-benzothiazole (0.1 g,0.31 mmol) was treated with boron tribromide under standard condition togive the title compound (30 mg, 33% yield) as a yellow solid. Mass spec:MH⁺=297.

Example 604-Bromo-6-methoxy-2-(2-isopropyl-3-methoxy-phenyl)-benzothiazole

The procedure was the same with4-bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole except using2-isopropyl-3-methoxy-benzoyl chloride instead of p-anisoyl chloride.Mass spec: MH⁺=392.

Example 614-Bromo-6-hydroxy-2-(2-isopropyl-3-hydroxy-phenyl)-benzothiazol

4-Bromo-6-methoxy-2-(2-isopropyl-3-methoxy-phenyl)-benzothiazole (60 mg,0.153 mmol) was treated with boron tribromide under standard conditionto give the title compound (30 mg, 54% yield) as a yellow solid. Massspec: MH⁺=364.

Example 62 4-Cyano-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Cyano-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (0.18 g, 0.61 mmol)was suspended in boron tribromide (1M in methylene chloride, 5.0 mL) andstirred at room temp under nitrogen for 18.0 hr. Reaction was pouredinto aqueous hydrochloric acid (1M). Solid was collected by filtrationand washed with water. Washed solid was further purified by flashchromatography on silica affording the title compound (150 mg, 92%) as ayellow solid. MH⁺=269; ¹H NMR (300 MHZ, DMSO-d⁶,) d 10.42(s, 1H),10.27(s, 1H), 7.91(d, J=8.7 hz, 2H), 7.77(d, J=2.4 hz, 1H), 7.36(d,J=2.4 hz, 1H), 6.94 (d, J=8.4 hz, 2H). The starting4-Cyano-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole was prepared asfollows:

a. N-(2-Bromo-4-Methoxy-phenyl)-4-methoxy-benzamideTo a solution containing 2-bromo-4-methoxy-aniline^(1.) (3.2 g, 15.8mmol) in pyridine (25 mL) was added p-anisoyl chloride (2.82 g, 16.5mmol) dropwise under nitrogen. The reaction was stirred at room temp for1.0 hr. Reaction was poured cautiously into aqueous 1M hydrochloric acidand extracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated brine and concentratedin vacuo. This tan solid was suspended in a solution containing:methanol (10 mL), methylene chloride (30 mL), and ethyl acetate (5 mL),mixed for 5 min, then allowed to sit for 15 min, yielding a white solid.This solid was collected by filtration and dried under vacuum yieldingthe title compound (1.97 g, 37%) as a white solid. Mass spec: MH⁺=336.^(1.)Prepared following literature procedure: Tet. (56) 2000, 1469

b. N-(2-Bromo-4-Methoxy-phenyl)-4-methoxy-thiobenzamideN-(2-Bromo-4-Methoxy-phenyl)-4-methoxy-benzamide (1.87 g, 5.6 mmol) andLawesson's reagent (1.35 g, 3.3 mmol) were suspended in chlorobenzene(15 mL) and heated to reflux under nitrogen for 3.0 hr. Reaction wascooled, solvent removed under vacuum yielding a yellow-orange solid.Solid was dissolved in ethyl acetate and washed with: 1) 1N HCl, 2)saturated brine. Remove solvent under vacuum. Solid was washed withhexane and dried under vacuum yielding the title compound (1.93 g, 98%)as an orange solid. Mass spec: MH⁺=352.

c. 4-Bromo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazoleN-(2-Bromo-4-Methoxy-phenyl)-4-methoxy-thiobenzamide (1.5 g, 4.25 mmol)was wetted with ethanol (5.0 mL). 30% Aqueous sodium hydroxide (10M, 3.4mL) was added and stirred for 5 min. Water (6.8 mL) was added to providea final suspension of 10% aqueous sodium hydroxide. Aliquots (1 mL) ofthis mixture were added at 1 min intervals to a heated (85° C.) stirredsolution containing potassium ferricyanide (5.6 g, 17 mmol) in water (50mL). Reaction was kept at 85° C. for 30 min, and then cooled to roomtemp. Cold water (120 mL) was added. Mixture was allowed to situndisturbed for 30 min. Precipitate was collected by filtration, washedwith water, and dried under vacuum. Solid was washed with ether thendried under vacuum at 35° C. yielding the title compound (1.2 g, 80%) asa pale tan solid. Mass spec: MH⁺=350.

d. 4-Iodo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole4-Bromo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (0.86 g, 2.46mmol), copper (1) iodide (2.34 g, 12.3 mmol), and potassium iodide (4.08g, 24.6 mmol) were suspended in DMSO (12 mL) and heated to 175° C. undernitrogen for 4 hr, then cooled to room temp. Reaction was pouredcautiously into aqueous hydrochloric acid (1.0M), and extracted withethyl acetate. Ethyl acetate extracts were washed with: 1) hydrochloricacid (1.0M), 2) saturated sodium thiosulfate, 3) saturated brine andconcentrated in vacuo. Solid was washed with methylene chloride/hexane(1:1), dried under vacuum yielding the title compound (0.87 g, 89%) as apale orange solid MH⁺=398.

e. 4-Cyano-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole4-Iodo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (258 mg, 0.65 mmol)and copper (1) cyanide (87 mg, 0.975 mmol) were suspended in DMF (6.0mL) under nitrogen and heated to 150° C. for 2.0 hr then cooled to roomtemp. Reaction was poured cautiously into aqueous 1M hydrochloric acidand extracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated brine and concentratedin vacuo. Solid was washed with ether/hexane (1:2), then dried undervacuum yielding the title compound (0.185 g, 96%) as a pale tan solid.MH⁺=297

Example 63 4-Bromo-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Bromo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (200 mg, 0.57 mmol)was suspended in boron tribromide (1M in methylene chloride, 7.5 mL) andstirred at room temp under nitrogen for 3.0 hr. Reaction was poured intoaqueous hydrochloric acid (1M) and extracted with ethyl acetate. Ethylacetate extracts were washed with: 1) hydrochloric acid (1.0M), 2)saturated brine and concentrated in vacuo. Washed solid was furtherpurified by flash chromatography on silica affording the title compound(184 mg, 100%) as a yellow solid. MH⁺=322

Example 64 4-Iodo-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Iodo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (175 mg, 0.44 mmol)[Compound of Example 1d] was suspended in boron tribromide (1M inmethylene chloride, 3.5 mL) and stirred at room temp under nitrogen for72.0 hr. Reaction was poured into aqueous hydrochloric acid (1M) andextracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated brine and concentratedin vacuo. Washed solid was further purified by flash chromatography onsilica affording the title compound (158 mg, 97%) as a yellow solid.MH⁺=370

Example 65 4-Chloro-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

4-Chloro-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (0.27 g, 0.88mmol) was suspended in boron tribromide (1M in methylene chloride, 7.0mL) and stirred at room temp under nitrogen for 18.0 hr. Reaction waspoured cautiously into aqueous 1M hydrochloric acid and extracted withethyl acetate. Ethyl acetate extracts were washed with: 1) hydrochloricacid (1.0M), 2) saturated brine and concentrated in vacuo. Solid waswashed with methylene chloride/methanol (97:3, 10 mL), and dried undervacuum yielding the title compound (0.240 g, 98%) as a tan solid. Massspec: MH⁺=278The starting 4-Chloro-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole wasprepared as follows:

a. N-(2-Chloro-4-hydroxy-phenyl)-4-methoxy-benzamideTo a solution containing 2-chloro-4-hydoxy-aniline hydrochloride(1.44 g,8 mmol) in pyridine (10 mL) was added p-anisoyl chloride (1.38 g, 8.1mmol) dropwise under nitrogen. The reaction was stirred at room temp for1.0 hr. Reaction was poured cautiously into aqueous 1M hydrochloric acidand extracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated brine and concentratedin vacuo. This solid was washed with ether and dried under vacuumyielding the title compound (1.68 g, 76%) as a tan solid. Mass spec:MH⁺=278.

b. N-(2-Chloro-4-methoxy-phenyl)-4-methoxy-benzamideN-(2-Chloro-4-hydroxy-phenyl)-4-methoxy-benzamide (1.83 g, 6.59 mmol)and potassium carbonate (1.82 g, 13.2 mmol) were suspended in DMF (15mL). Methyl iodide (0.62 mL, 9.89 mmol) was added and stirred at roomtemp under nitrogen for 15 min, then heated to 95° C. for 18 hr.Reaction was cooled to room temp then poured cautiously into aqueous 1Mhydrochloric acid and extracted with ethyl acetate. Ethyl acetateextracts were washed with: 1) hydrochloric acid (1.0M), 2) saturatedNaHCO₃, 3) saturated brine and concentrated in vacuo. Washed solid wasfurther purified by flash chromatography on silica affording the titlecompound (1.31 g, 68%) as a white solid. MH⁺=292

c. N-(2-Chloro-4-methoxy-phenyl)-4-methoxy-thiobenzamideN-(2-Chloro-4-methoxy-phenyl)-4-methoxy-benzamide (0.62 g, 2.13 mmol)and Lawesson's reagent (0.52 g, 1.28 mmol) were suspended inchlorobenzene (10 mL) and heated to reflux under nitrogen for 3.0 hr.Reaction was cooled, solvent removed under vacuum yielding ayellow-orange solid which was further purified by flash chromatographyon silica affording the title compound (0.60 g, 92%) as a yellow solid.Mass spec: MH⁺=308

d. 4-Chloro-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazoleN-(2-Chloro-4-Methoxy-phenyl)-4-methoxy-thiobenzamide (0.307 g, 1 mmol)was wetted with ethanol (4.0 mL). 30% Aqueous sodium hydroxide (10M, 0.8mL) was added and stirred for 5 min. Water (2.4 mL) was added to providea final suspension of 10% aqueous sodium hydroxide. Aliquots (1 mL) ofthis mixture were added at 1 min intervals to a heated (85° C.) stirredsolution containing potassium ferricyanide (1.32 g, 4 mmol) in water (20mL). Reaction was kept at 85° C. for 30 min, and then cooled to roomtemp. Reaction was poured cautiously into aqueous 1M hydrochloric acidand extracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated NaHCO₃, 3) saturatedbrine and concentrated in vacuo. Solid was washed with ether, driedunder vacuum yielding the title compound (0.285 g, 93%) as a whitesolid. Mass spec: MH⁺=306

Example 66 2-(4-Hydroxy-phenyl)-4-Trifluormethyl-6-hydroxy-benzothiazole

6-Methoxy-2-(4-Methoxy-phenyl) 4-Trifluoromethyl--benzothiazole (0.08 g,0.23 mmol) was suspended in boron tribromide (1M in methylene chloride,5.0 mL) and stirred at room temp under nitrogen for 18.0 hr. Reactionwas poured into aqueous hydrochloric acid (1M). Solid was collected byfiltration and washed with water. Washed solid was further purified byflash chromatography on silica affording the title compound (21 mg, 29%)as a white solid. MH⁺=311The starting 6-Methoxy-2-(4-Methoxy-phenyl)4-Trifluoromethyl-benzothiazole was prepared as follows:

a. 6-Methoxy-2-(4-Methoxy-phenyl) 4-Trifluoromethyl-benzothiazole4-Iodo-6-methoxy-2-(4-Methoxy-phenyl)-benzothiazole (397 mg, 1.0 mmol)[Compound of Example 1d] and copper (0) powder (150 mg, 2.36 mmol) weresuspended in pyridine (15.0 mL) under nitrogen in a Parr bomb equippedwith gas inlet. Trifluoromethyl Iodide (6.0 g, 30.6 mmol)was added viathe gas inlet. Reaction was heated to 165° C. for 48.0 hr then cooled toroom temp. Remove pyridine under vacuum Reaction was suspended in ethylacetate/1MHCl (200 mL, 1: 1), and filtered. The ethyl acetate extractwas washed with: 1) hydrochloric acid (1.0M), 2) saturated NaHCO₃ 3)saturated sodium thiosulfate, 4) saturated brine and concentrated invacuo. Residue was further purified by flash chromatography on silicaaffording the title compound (0.16 g, 47%) as a tan solid. Mass spec:MH⁺=339

Example 67 2-(4-Hydroxy-phenyl)-6-hydroxy-benzothiazole

6-Methoxy-2-(4-methoxy-phenyl)-benzothiazole (134 mg, 0.49 mmol) andpyridine hydrochloride ( 1.34 g, 11.6 mmol) were heated to 200° C. undernitrogen for 40 min, and then cooled to room temp. Reaction was pouredcautiously into aqueous hydrochloric acid (1M) and extracted with ethylacetate. Ethyl acetate extracts were washed with: 1) hydrochloric acid(1.0M), 2) saturated brine and concentrated in vacuo. Residue was washedwith ether/hexane (1:4), dried under vacuum yielding the title compound(119 mg, 100%) as a yellow solid. MH⁺=244The starting 6-Methoxy-2-(4-methoxy-phenyl)-benzothiazole was preparedas follows:

a. 2-Bromo-6-methoxy-benzothiazoleTo a solution containing dry Copper (11) bromide (2.68 g, 12 mmol),tri(ethylene glycol) dimethyl ether (5 g) in acetonitrile (150 mL) wasadded isoamyl nitrite (2 mL, 15 mmol).Reaction was stirred at room temp under nitrogen for 30 min. To thissuspension was added, dropwise, a solution (obtained by sonification)containing 2-amino-6-methoxy-benzothiazole (1.8 g, 10 mmol) andtri(ethylene glycol) dimethyl ether (5 g) in acetonitrile (50 mL).Reaction was stirred at room temp for 10 min, and then heated to 50° C.for 3 hr. Reaction was cooled to room temp, poured cautiously intoaqueous 6M hydrochloric acid and extracted with ethyl acetate. Ethylacetate extracts were washed with: 1) hydrochloric acid (1.0M), 2)saturated brine and concentrated in vacuo. Residue was crystallized fromether/hexane (1:10) yielding the title compound (1.48 g, 61%) MH⁺=244Supernatant solution was concentrated, dried under vacuum yielding2,7-dibromo-6-methoxy-benzothiazole (0.45 g, 14%) as a yellow solid.MH⁺=322

b. 6-Methoxy-2-(4-methoxy-phenyl)-benzothiazole2-Bromo-6-methoxy-benzothiazole (244 mg, 1 mmol),4-methoxy-phenyl-boronic acid (198 mg, 1.3 mmol),tetrakis(triphenylphosphine) palladium (0) (58 mg, 0.05 mmol), andcesium fluoride (380 mg, 2.5 mmol) were suspended in acetonitrile (10mL) and heated to reflux for 90 min under nitrogen. Reaction was cooledto room temp, poured cautiously into aqueous 1M hydrochloric acid andextracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated NaHCO₃, 3) saturatedbrine and concentrated in vacuo. Washed solid was further purified byflash chromatography on silica affording the title compound (271 mg,100%) as a pale yellow solid. MH⁺=272

Example 68 7-Chloro-6-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

7-Bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole (65 mg, 0.186mmol)) and pyridine hydrochloride (1.6 g, 13.8 mmol) were heated to 200°C. under nitrogen for 45 min, and then cooled to room temp. Reaction waspoured cautiously into aqueous 1M hydrochloric acid and extracted withethyl acetate. Ethyl acetate extracts were washed with: 1) hydrochloricacid (1.0M), 2) saturated brine and concentrated in vacuo. Residue wasfurther purified by flash chromatography on silica affording the titlecompound (51 mg, 99%) as a white solid. MH⁺=278The starting 7-Bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole wasprepared as follows:

a. 7-Bromo-6-methoxy-2-(4-methoxy-phenyl)-benzothiazole2,7-Dibromo-6-methoxy-benzothiazole (94 mg, 0.29 mmol),4-methoxy-phenyl-boronic acid (47 mg, 0.31 mmol),tetrakis(triphenylphosphine) palladium (0) (19 mg, 0.015 mmol), andcesium fluoride (110 mg, 0.725 mmol) were suspended in acetonitrile (10mL) and heated to reflux for 90 min under nitrogen. Reaction was cooledto room temp, poured cautiously into aqueous 1M hydrochloric acid andextracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated NaHCO₃, 3) saturatedbrine and concentrated in vacuo. Washed solid was further purified byflash chromatography on silica affording the title compound (71 mg, 70%)as a white solid. MH⁺=350

Example 69 7-Cyano-5-hydroxy-2-(4-hydroxy-phenyl)-benzothiazole

5-Methoxy-2-(4-methoxy-phenyl)-benzothiazole-7-carbonitrile (0.04 g,0.135 mmol) was suspended in boron tribromide (1M in methylene chloride,5.0 mL) and stirred at room temp, under nitrogen for 48 hr. Reaction waspoured into aqueous hydrochloric acid (1M) and extracted with ethylacetate. Ethyl acetate extracts were washed with: 1) hydrochloric acid(1.0M), 2) saturated NaHCO₃, 3) saturated brine and concentrated invacuo. This material was further purified by chromatography on silicayielding the title compound (14 mg, 39%) as a tan solid. Mass spec:MH⁺=296

5-Methoxy-2-(4-hydroxy-phenyl)-benzothiazole-7-carbonitrile (7 mg, 18%)was also obtained as a white solid from this chromatography.

The starting 7-Cyano-5-Methoxy-2-(4-methoxy-phenyl)-benzothiazole wasprepared as follows:

a. 3-Amino-5-methoxy-phenol3,5 Dimethoxy-aniline (1.53 g, 10 mmol) and pyridine hydrochloride (6.9g, 60 mmol) were heated to 190° C. under nitrogen for 60 min, and thencooled to room temp. Reaction was poured cautiously into saturatedNaHCO₃ and extracted with ethyl acetate. Ethyl acetate extracts werewashed with: 1) saturated NaHCO₃, 2) saturated brine and concentrated invacuo. Residue was further purified by flash chromatography on silicaaffording the title compound (600 mg, 44%) as a tan oil. MH⁺=139

b. N-(3-Hydroxy-5-Methoxy-phenyl)-4-methoxy-benzamideTo a solution containing 3-Amino-5-methoxy-phenol (0.59 g, 4.28 mmol) inpyridine (5 mL) was added p-anisoyl chloride (0.77 g, 4.49 mmol)dropwise under nitrogen. The reaction was stirred at room temp for 18hr. Reaction was cautiously poured into aqueous 1M hydrochloric acid andextracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) hydrochloric acid (1.0M), 2) saturated brine and concentratedin vacuo. This tan solid was further purified by chromatography onsilica yielding the title compound (0.97 g, 83%) as a tan solid. Massspec: MH⁺=274.

c. Trifluoro-methanesulfonic-acid 3-methoxy-5-(4-methoxy-benzoylamino)-phenyl ester To a chilled (0° C.) solution containingN-(3-Hydroxy-5-Methoxy-phenyl)-4-methoxy-benzamide (0.546 g, 2 mmol),diisopropylethyl amine (646 mg, 5 mmol) in methylene chloride (15 mL)was added, dropwise, a solution containing triflic anhydride (0.846 g, 3mmol) in methylene chloride (6 mL) under nitrogen. The reaction wasstirred at 0° C. for 10 min and then allowed to warm to room temp for 18hr. Reaction was poured cautiously into saturated NaHCO₃ and extractedwith ethyl acetate. Ethyl acetate extracts were washed with: 1)saturated NaHCO₃, 2) saturated brine and concentrated in vacuo. Thismaterial was further purified by chromatography on silica yielding thetitle compound (0.44 g, 54%) as a tan oil. Mass spec: MH⁺=406.

d. N-(3-Cyano-5-methoxy-phenyl)-4-methoxy-benzamideTrifluoro-methanesulfonic-acid 3-methoxy-5-(4-methoxy-benzoylamino)-phenyl ester (0.41 g, 1 mmol) and potassium cyanide (0.163 g, 2.5mmol) were suspended in DMF (5 ml) and heated to 120° C. under nitrogenfor 18 hr. Reaction was cooled to room temp, poured cautiously intosaturated NaHCO₃ and extracted with ethyl acetate. Ethyl acetateextracts were washed with: 1) saturated NaHCO₃, 2) hydrochloric acid(1M), 3) saturated brine and concentrated in vacuo. This material wasfurther purified by chromatography on silica yielding the title compound(0.17 g, 60%) as a tan solid. Mass spec: MH⁺=283.

e. N-(3-Cyano-5-methoxy-phenyl)-4-methoxy-thiobenzamideN-(3-Cyano-5-methoxy-phenyl)-4-methoxy-benzamide (80 mg, 0.28 mmol) andLawesson's reagent (69 mg, 0.17 mmol) were suspended in chlorobenzene (5mL) and heated to reflux under nitrogen for 3.0 hr. Reaction was cooled,solvent removed under vacuum. Solid was dissolved in ethyl acetate andwashed with: 1) hydrochloric acid (1.0M), 2) saturated NaHCO₃, 3)saturated brine and concentrated in vacuo yielding the title compound(83 mg, 100%) as a yellow-orange solid. Mass spec: MH⁺=298

f. 7-Cyano-5-Methoxy-2-(4-methoxy-phenyl)-benzothiazoleN-(3-Cyano-5-methoxy-phenyl)-4-methoxy-thiobenzamide (90 mg, 0.30 mmol)was wetted with ethanol (3.0 mL). 30% Aqueous sodium hydroxide (10M, 2.4mL) was added and stirred for 5 min. Water (4.8 mL) was added to providea final suspension of 10% aqueous sodium hydroxide. Aliquots (1 mL) ofthis mixture were added at 1 min intervals to a heated (85° C.) stirredsolution containing potassium ferricyanide (398 mg, 1.21 mmol) in water(6 mL). Reaction was kept at 85° C. for 30 min, and then cooled to roomtemp. Cold water (120 mL) was added. Extract with ethyl acetate. Ethylacetate extracts were washed with: 1) hydrochloric acid (1.0M), 2)saturated NaHCO₃, 3) saturated brine and concentrated in vacuo. Thismaterial was further purified by chromatography on silica yielding thetitle compound (44 mg, 49%) as a white solid. Mass spec: MH⁺=296.

Example 70 2-(4-Amino-phenyl)-6-hydroxy-benzothiazole

4-(6-Methoxy-benzothiazol-2-yl)-phenylamine (27 mg, 0.105 mmol) wassuspended in boron tribromide (1M in methylene chloride, 3.0 mL) andstirred at room temp under nitrogen for 18.0 h. Reaction was poured intosaturated NaHCO₃ and extracted with ethyl acetate. Ethyl acetateextracts were washed with: 1) saturated NaHCO₃, 2) saturated brine andconcentrated in vacuo. This material was further purified bychromatography on silica yielding the title compound (25 mg, 100%) as atan solid. Mass spec: MH⁺=243The starting 4-(6-Methoxy-benzothiazol-2-yl)-phenylamine was prepared asfollows:

2-Bromo-6-methoxy-benzothiazole (244 mg, 1 mmol) [Example 67],4-(4,4,5,5-tetramethyl-1,3,2-dioxboralan-2-yl)-aniline (285 mg, 1.3mmol), tetrakis(triphenylphosphine) palladium (0) (58 mg, 0.05 mmol),and cesium fluoride (380 mg, 2.5 mmol) were suspended in acetonitrile(10 mL) and heated to reflux for 90 min under nitrogen. Reaction wascooled to room temp, poured cautiously into saturated NaHCO₃ andextracted with ethyl acetate. Ethyl acetate extracts were washedwith: 1) saturated NaHCO₃, 2) saturated brine and concentrated in vacuo.Washed solid was further purified by flash chromatography on silicaaffording the title compound (190 mg, 74%) as a pale yellow solid.MH⁺=272

Example 71 6-Bromo-2-(4-hydroxy-phenyl)-benzothiazole

6-Bromo-2-(4-methoxy-phenyl)-benzothiazole (120 mg, 0.375 mmol) wassuspended in boron tribromide (1M in methylene chloride, 7.0 mL) andstirred at room temp under nitrogen for 18.0 h. Reaction was poured intosaturated brine and extracted with ethyl acetate. Ethyl acetate extractswere washed with: 1) hydrochloric acid (1.0M), 2) saturated brine andconcentrated in vacuo. Residue was washed with hexane, and dried undervacuum yielding the title compound (115 mg, 100%) as a tan solid. Massspec: MH⁺=306The starting 6-Bromo-2-(4-methoxy-phenyl)-benzothiazole was prepared asfollows:

a. N-(4-Bromo-phenyl)-4-methoxy-benzamideTo a solution containing 4-bromo-aniline (1.0 g, 7 mmol) in pyridine (7mL) was added p-anisoyl chloride (0.77 mL, 7.1 mmol) dropwise undernitrogen. The reaction was stirred at room temp for 30 min. Reaction waspoured cautiously into saturated NaHCO₃ and extracted with ethylacetate. Ethyl acetate extracts were washed with: 1) saturated NaHCO₃,2) saturated brine and concentrated in vacuo. This solid was washed witha solution containing: ether/ hexane (1:5, 10 mL), dried under vacuum,yielding the title compound (1.97 g, 92%) as a white solid. Mass spec:MH⁺=306.

b. N-(4-Bromo-phenyl)-4-methoxy-thiobenzamideN-(4-Bromo-phenyl)-4-methoxy-benzamide (1.95 g, 6.37 mmol) andLawesson's reagent (1.55 g, 3.82 mmol) were suspended in chlorobenzene(25 mL) and heated to reflux under nitrogen for 3.0 h. Reaction wascooled, solvent removed under vacuum. Solid was dissolved in ethylacetate and washed with: 1) hydrochloric acid (1.0M), 2) saturated brineand concentrated in vacuo. Residue was further purified bychromatography on silica yielding the title compound (1.85 g, 90%) as ayellow-orange solid. Mass spec: MH⁺=322

c. 6-Bromo-2-(4-methoxy-phenyl)-benzothiazoleN-(4-Bromo-phenyl)-4-methoxy-thiobenzamide (483 mg, 1.5 mmol) was wettedwith ethanol (4.0 mL). 30% Aqueous sodium hydroxide (10M, 1.2 mL) wasadded and stirred for 5 min. Water (2.4 mL) was added to provide a finalsuspension of 10% aqueous sodium hydroxide. Aliquots (1 mL) of thismixture were added at 1 min intervals to a heated (85° C.) stirredsolution containing potassium ferricyanide (1.98 g, 6 mmol) in water (25mL). Reaction was kept at 85° C. for 30 min, and then cooled to roomtemp. Cold water (200 mL) was added. Mixture was allowed to situndisturbed for 30 min. Precipitate was collected by filtration, washedwith water, and dried under vacuum. Solid was dried under vacuum at 37°C. yielding the title compound (0.45, 93%) as a pale yellow solid. Massspec: MH⁺=320.

1-17. (canceled)
 18. A compound of the formula (I)

or a pharmaceutically acceptable salt thereof, wherein: X is S; R¹ isC₁₋₈alkyl, phenyl or a 5- or 6-membered ring heterocycle containing 1, 2or 3 heteroatoms each independently selected from O, N and S andadditionally having 0 or 1 oxo groups and 0 or 1 fused benzo rings,wherein the C₁₋₈alkyl or heterocycle is substituted by 0, 1, 2 or 3substituents selected from —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a),—CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), S(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl and the phenyl issubstituted by 0, 1, 2 or 3 substituents selected from —R^(a),—CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O) R^(a),—NR^(a)S(═O) R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; R³ is —R^(a),—OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; or R³ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro; R⁴ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a),—CO2R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro or C₁₋₃haloalkyl; R⁵ is —R^(a),—OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; R⁶ is —R^(a), —OR^(a), —SR^(a),—NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and or C₁₋₃haloalkyl;or R⁶ is C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano and nitro; and R^(a) is H,C₁₋₆alkyl, C₁₋₃haloalkyl, phenyl or benzyl; with the provisos that whenthe compound is in free base form and: a) R¹ is phenyl, the benzene ringof the benzthiazole is not substituted by 4-methoxy, 5,6-dimethoxy,6-hydroxy or 6-methoxy; and b) R¹ is 4-methylphenyl, the benzene ring ofthe benzthiazole is not unsubstituted and is not substituted by 4-, 5-or 6-fluoro, 4-, 6- or 7-methoxy, 5-chloro, 4-, 5-, 6- or 7-hydroxy, 4-,5-, 6- or 7-acetoxy or 6-nitro; and R¹ is not 4-chloro- or4-fluorophenyl when the benzene ring of the benzthiazole is substitutedby 5-hydroxy or 5-mercapto.
 19. (canceled)
 20. (canceled)
 21. Thecompound of the formula (I) or a pharmaceutically acceptable saltthereof according to claim 18 wherein R¹ is unsubstituted or substitutedphenyl.
 22. The compound of the formula (I) or a pharmaceuticallyacceptable salt thereof according to claim 18 wherein R¹ is anunsubstituted or substituted 5- or 6-membered ring heterocycle.
 23. Thecompound of the formula (I) or a pharmaceutically acceptable saltthereof according to claim 18 wherein R¹ is halophenyl, C₁₋₄alkylphenyl,cyanophenyl or trifluoromethylphenyl.
 24. The compound of the formula(I) or a pharmaceutically acceptable salt thereof according to claim 18wherein R³ is halo, cyano, carbamoyl or C₁₋₆alkyl.
 25. The compound ofthe formula (I) or a pharmaceutically acceptable salt thereof accordingto claim 18 wherein R³ is hydrogen.
 26. The compound of the formula (I)or a pharmaceutically acceptable salt thereof according to claim 18wherein R⁴ is halo, hydroxy or C₁₋₆alkoxy.
 27. The compound of theformula (I) or a pharmaceutically acceptable salt thereof according toclaim 18 wherein R⁴ is hydrogen.
 28. The compound of the formula (I) ora pharmaceutically acceptable salt thereof according to claim 18 whereinR⁵ is halo, hydroxy or C₁₋₆alkoxy.
 29. The compound of the formula (I)or a pharmaceutically acceptable salt thereof according to claim 18wherein R⁵ is hydrogen.
 30. The compound of the formula (I) or apharmaceutically acceptable salt thereof according to claim 18 whereinR⁶ is halo, C₁₋₄alkyl, trifluoromethyl, hydroxy, C₁₋₄alkoxy, carboxy,C₁₋₄alkoxycarbonyl, cyano, halomethyl, cyanoC₁₋₄alkyl, carbamoyl,methylcarbamoyl or dimethylcarbamoyl.
 31. The compound of the formula(I) or a pharmaceutically acceptable salt thereof according to claim 18wherein R⁶ is hydrogen.
 32. (canceled)
 33. The compound of the formula(I) or a pharmaceutically acceptable salt thereof according to claim 18wherein R⁶ is cyano or carboxy; R⁴ is hydroxy; and R³ and R⁵ are bothhydrogen.
 34. A pharmaceutical composition comprising a compound of theformula (I) or a pharmaceutically acceptable salt thereof as defined inclaim 18 and a pharmaceutically acceptable carrier.
 35. (canceled)
 36. Amethod for achieving selective targeting of the β-estrogen receptor,which comprises administering an effective amount of a compound of theformula (I) or a pharmaceutically acceptable salt thereof according toclaim
 18. 37. A method for treating Alzheimer's disease, anxietydisorders, depressive disorders, osteoporosis, cardiovascular disease,rheumatoid arthritis or prostate cancer, which comprises administeringan effective amount of a compound of the formula (I) or apharmaceutically acceptable salt thereof according to claim 18 to apatient in need of such treatment.
 38. (canceled)
 39. A process forpreparing a compound of the formula (I) or a pharmaceutically acceptablesalt thereof, as defined in claim 18 which comprises: a) cyclizing acompound of the formula:

wherein X, R¹, and R³-R⁶ are as defined in claim 18 and L is hydrogen ora leaving group; or b) cyclizing a compound of the formula:

wherein R¹, R³-R⁶ and X are as defined in claim 18 and L is hydrogen ora leaving group; or c) cyclizing a compound of the formula:

wherein R¹ and R³-R⁶ are as defined in claim 18; and thereafter,desired, forming a pharmaceutically acceptable salt.
 40. A method fortreating a condition for which ERT has a beneficial effect, whichcomprises administering an effective amount of a compound of the formula(I) or a pharmaceutically acceptable salt thereof according to claim 18to a patient in need of such treatment.